Beneficial effects of bio-fabricated selenium nanoparticles as seed nanopriming agent on seed germination in rice (Oryza sativa L.)

Climate change and increasing population pressure have put the agriculture sector in an arduous situation. With increasing demand for agricultural production overuse of inputs have accentuated the negative impact on environment. Hence, sustainable agriculture is gaining prominence in recent times with an emphasis on judicious and optimum use of resources. The field of nanotechnology can immensely help in achieving sustainability in agriculture at various levels. Use of nutrients and plant protection chemicals in nano-form can increase their efficacy even at reduced doses thus decreasing their pernicious impact. Seed priming is one of the important agronomic practices with widely reported positive impacts on germination, seedling growth and pathogen resistance. In the current study, the effect and efficacy of selenium nanoparticles synthesized using phyto-extracts as a seed priming agent is studied. This nanopriming enhanced the germination, hastened the seedling emergence and growth with an increase in seedling vigour and nutrient status. This eco-friendly and economical method of synthesizing nanoparticles of various nutrient minerals can optimize the resource use thus helping in sustainable agriculture by reducing environment damage without compromising on efficacy.


Characterization of green SeNPs
UV-Vis absorption spectra of extract, Na2SeO3, SeNPs were recorded with the help of UV-vis spectrophotometer by recording the wavelength range between 300-600 nm.The Raman spectroscopy was conducted at room temperature at 633 nm line of laser in the spectral range of 100 -500 cm −1 with the acquisition interval of 5 seconds via LabRAM HR Evol (HORIBA Scientific, USA).The FT-IR spectra of the SeNPs, was determined using the Spectrum3 FT-IR spectrophotometer (Perkin-Elmer, USA) in transmission mode.X-ray diffraction (XRD) analysis was performed on high Resolution X-ray diffractometer.The pattern for XRD spectra was recorded at 2θ = 10-80• by using Cu Kα radiation (λ= 1.540593 Å) at 20 kV.
The particle size and shape of SeNPs was revealed by Field Emission Scanning Electron Microscopy (FE-SEM) examination (EVO SEM MA 15/18, Carl-Zeiss, Germany).The energy dispersive X-ray spectroscopy (EDX) was performed using a Bruker EDX spectrometer to determine the elemental composition of the SeNPs.

Preparation of priming solution and seed priming method
The green synthesized SeNPs were used for seed priming and germination test.Different concentrations of SeNPs (20µM and 25µM) were prepared by dispersing NPs in distilled water via sonication for 40 min, designated as SeNP 20 and SeNP 25 respectively, followed by preparation of two concentrations Na2SeO3 (10µM and 20µM) by dissolving salt in distilled water, designated as Se 10 and Se 20 respectively.The concentrations were earlier optimized by screening for concentration gradient in a control plant.Healthy and uniform rice (Oryza sativa L.) seeds were selected and were first with running tap water Subsequently seeds were surface sterilized with 2% of sodium hypochlorite for 10 min then washed with distilled water several times.Sterilized seeds were then soaked in different concentrations of T2-Se 10, T3-Se 20, T5-SeNP 20 and T5-SeNP 25 solution respectively for 24 h, and surface-dried on paper towel.Seeds were dried back to their original moisture content at room temperature (25 ± 2 °C), sealed in polythene bags and stored at 4 °C until further use.Seeds soaked in distilled water, were defined as hydropriming and designated as control.

Germination test, Seedling vigor and Biochemical assay
All the tests on germination were performed in triplicates.Whatman filter paper was placed on sterilized Petri plates.Ten primed seeds of each treatment were placed in their respective labelled Petri plate, and then moistened with 5 mL of distilled water.
All the Petri plates were kept in an incubator under dark condition at 30 ○ in the BOD.After 24 h of incubation, germination tests for, amylase activity, total soluble sugar content and starch content were performed and repeated upto 96 h at the interval of 24 h.Simultaneously, the seeds were allowed to germinate and the germination was monitored for next 7 days till 100% germination achieved in control.When the radicle length of germinating seeds extended up to 5 mm in length then it was considered as germinated and data on germination percentage (G%) was calculated according to the previous literatures (Ellis and Roberts, 1981;Feizi et al., 2013).

2,3,5-Triphenyltetrazolium Chloride (TTC) Staining
TTC staining is a frequently used method to determine the viability of cells or tissues.TTC is a white salt and substrate of dehydrogenases and its metabolite is a red dye, which turns the cell red in viable tissue and white in dead tissue.After incubation in water, SeNPs and Se salt for 24, 48, 72 and 96 h five whole seeds with embryo were selected from each treatment and stained with 0.5% TTC at 35 о C for 3 h and then washed five times with distilled water and photographed under a SteREOLumarV12 stereomicroscope (R. Li et al., 2017;Soares et al., 2016).

Starch metabolism and starch agar plate α-amylase assay
After germination starch metabolism in germinating seeds were determined by measuring α-amylase activity, total soluble sugar content and starch content upto 96 h at the interval 24 h.The activity of αamylase enzyme was assayed from the saccharifying activity as per the protocol described by Bernfeld (1955).The enzyme activity calculated by the formula given by Yaldagard et al. (2008).For confirming the effect nanopriming on α-amylase activity, starch-agar plate assay (Qualitative assessment) was performed following the protocol of Chen et al. (2006).The embryo-less half-seeds were sterilized and placed perpendicularly on starch-agar plates [2% agar plate containing 0.2 % soluble potato starch, 10 mM sodium acetate and 2 mM CaCl2 (pH 5.3)] and incubated at room temperature in the dark for and observed for 3 days.1 μM gibberellic acid (GA3) was added to cooled agar that acted as positive control (GA+), while plate having no GA3 was defined as negative control (GA-).After 3 days of incubation, embryo-less seeds were removed and staining agar plate with IKI solution for testing the α-amylase production.
Clear zones (transparent halos) appear if α-amylases are synthesized in endosperms and secreted into the starch agar, resulting in starch hydrolysis.The experiment was performed in triplicates.Total soluble sugars (TSS) were estimated using anthrone reagent as per the procedure outlined by Dubois et al. (1956).Starch content was estimated using the method described by Hodge and Hofreiter (1962).The amount of sugar was determined using the standard curve prepared from glucose.

Antioxidant enzyme assay
Nitroblue tetrazolium (NBT) photochemical assay was used to estimate the activity of superoxide dismutase (SOD) (Beyer and Fridovich, 1987).After 24 h of imbibition, 500 mg seeds were grinded and mixed with 100 mM potassium phosphate buffer (PPB) (pH 7.8) containing 1 % polyvinyl-pyrrolidone (PVP), 0.1 mM ethylene-diamine-tetra acetic acid (EDTA) and 0.5 % Triton X-100.Followed by centrifugation at 35,000 g for 15 min at 4 •C and supernatant was collected.The resultant supernatant was used to detect antioxidant enzyme activity.For SOD, 500 μL of supernatant was mixed with 4 mL solution containing 20 mM methionine, 0.15 mM EDTA and 0.12 mM NBT.Exposure to fluorescent lamps for 30 min was given to tubes containing samples whereas the sample labelled as blank was kept in the dark.One unit of the enzyme activity was defined as the quantity of enzyme required to result in a 50 % inhibition of the rate of NBT reduction at 560 nm.Activity of catalase (CAT) was anticipated in terms of the dissociation of H2O2 at 240 nm for 1 min (extinction coefficient of 39.4 mM 1 cm 1) according to Aebi (1984).
2 mL of reaction mixture contained 200 μL enzyme extract, 50 mM PPB(pH 7.0), 10 mM H2O2 and 1 mM EDTA. 1 n mol H2O2 dissociated min 1 defined as reactivity of one unit of enzyme.Activity of ascorbate peroxidase (APX) was estimated according to Chen and Asada (1989).Decreased absorbance was monitored at 290 nm over a period of 4 min.
For this the reaction mixture comprised 500 μL of enzyme extract, 50 mM PPB (pH 7.0), 0.5 mM ascorbate and 0.2 mM H2O2.The activity was calculated using an extinction coefficient of 2.8 mM-1 cm-1.

Histochemical localization in seeds
The accumulation of ROS (H2O2 and O2 -) were localized using 3,3-diaminobenzidine (DAB) and nitro-blue tetrazolium (NBT) respectively whereas the production and accumulation of •OH, can indirectly reflect the peroxidase activity which was detected histochemically by TMB staining as described by (Chen et al.,2016 andLi et al., 2017b) in seeds.For H2O2 and O2 -five Seeds from each treatments being soaked for different intervals (24, 48 h and 72 h) were collected and incubated again in 1 mg ml-1 of DAB stain solution and 1 mM NBT in 10 mM Tris-HCl respectively for 30 min at room temperature.For TMB staining seeds soaked for different intervals (24, 48 h and 72 h) from each treatment were selected and incubated in 0.2% (w/v) TMB and 1 mM H2O2 in 20 mM phosphate buffer (pH 6.5) at room temperature for 30 min.Finally all the stained seeds were photographed with the SteREOLumarV12 stereomicroscope.

Statistical Analysis
The data obtained from four different replicates are analysed for Analysis of variance (ANOVA), Duncan's multiple range test (DMRT) post-hoc analysis and correlation analysis by using software R (v 4.2.1).UV, XRD, Raman and FT-IR graphs were generated using Origin v2022b.Other graphs are generated using Graphpad Prism (v8.0.2).